16,17-carbocyclic condensed steroid compounds having selective estrogenic activity

ABSTRACT

The invention discloses a steroid compound having the formula (1), wherein dotted bonds represent optional double bonds; R6 is H, ═CH2, or —CH3, or —CH2—CH3; R7 is H, C1-4-alkyl, C2-5 alkenyl or C2-5-alkynyl, wherein the alkyl, alkenyl or alkynyl group may be substituted with 1 to 3 halogen atoms independently chosen from the group of fluorine or chlorine atoms; R11 is H, C1-4-alkyl, C2-4-alkenyl, C2-4-alkynyl or C1-4-alkylidene, wherein the alkyl, alkenyl, alkynyl or alkylidene group may be substituted with 1-3 halogen atoms independently chosen from the group of fluorine or chlorine atoms; E represents together with carbon atoms 16 and 17 of the steroid skeleton a four to seven-membered ring, said ring being α and in cis-configuration with respect to the steroid skeleton, optionally comprising one or two endocyclic bonds; or a prodrug thereof. Such compounds can be used in therapy and for methods for selective modification of the activity of estrogen receptors.

FIELD OF THE INVENTION

The present invention relates to steroid compounds with an additional Ering connected to ring D of the steroid skeleton having estrogenicactivity.

BACKGROUND OF THE INVENTION

There is continued interest in new compounds with affinity for theestrogen receptor. This stems from the discovery of two distinctsubtypes of receptors, denoted ERα and ERβ (see Mosselman et al., FEBSLetters 392 (1996) 49-53 as well as EP-A-0 798 378). Compounds which areselective for such subtypes of receptors make it possible to provide amore selective estrogen-receptor related treatment. Advantages can forexample be obtained from the different distribution of receptor subtypesin human tissue. This enables treatments with a lower burden ofestrogen-related side-effects. Examples of estrogen-related medicaltreatments which can benefit from selective compounds are those forcontraception, for therapy of menopausal complaints, osteoporosis, andestrogen dependent tumour control.

DETAILED DESCRIPTION OF THE INVENTION

In EP 0 869 132 estrogenic steroid compounds are described which havethe formula (1):

wherein:

-   -   dotted bonds represent optional double bonds;    -   R₆ is H, ═CH₂, or —CH₃, or —CH₂—CH₃;    -   R₇ is H, C₁₋₄-alkyl, C₂₋₅ alkenyl or C₂₋₅-alkynyl, wherein the        alkyl, alkenyl or alkynyl group may be substituted with 1 to 3        halogen atoms independently chosen from the group of fluorine or        chlorine atoms;    -   R₁₁ is H, C₁₋₄-alkyl, C₂₋₄-alkenyl, C₂₋₄-alkynyl or        C₁₋₄-alkylidene, wherein the alkyl, alkenyl, alkynyl or        alkylidene group may be substituted with 1 to 3 halogen atoms        independently chosen from the group of fluorine or chlorine        atoms;    -   E represents together with carbon atoms 16 and 17 of the steroid        skeleton a four to seven-membered ring, said ring being α in        cis-configuration with respect to the steroid skeleton,        optionally comprising one or two endocyclic bonds and        substituted with R_(E), which has a variety of meanings.

It was further disclosed in EP 0 869 132 that any alkyl, alkenyl,alkynyl and alkylidene group in the steroid compound having the formula(I) may be branched or unbranched. If R₆ or R₁₁ is connected to thesteroid skeleton through a single bond, the substituted carbon atom ofthe steroid skeleton either comprises a hydrogen atom or is involved ina double carbon-carbon bond. Compounds may contain different centres ofchirality and can exist as enantiomers and diastereomers. Hydroxylgroups may be capped with substituents such as acyl or alkyl leading toprodrugs of a compound according to formula 1.

It is now found that a steroid compound having formula 1 with symbolsand terms having the meanings as defined above, characterised in thatR_(E) is a β-hydroxy group, or a prodrug thereof, has, unexpectedly, aconsistently better selectivity for the estrogen receptor α combinedwith a high estrogen α potency. Such a compound, hereafter referred toas a compound of the invention, usually is not only very weakly activeon the estrogen receptor β, but in fact is generally an antagonist onthe estrogen receptor β, which contributes to the very high selectivityfor the estrogen receptor α and furthermore enables a selectivetreatment based on blockade of the estrogen receptor β. The compounds ofthis invention include the aforementioned enantiomers and diastereomerswithin its scope and each of the individual (R) and (S) enantiomers,substantially free, i.e. associated with less than 5%, preferably lessthan 2%, in particular less than 1% of the other enantiomer and mixturesof such enantiomers in any proportions including racemic mixturescontaining substantially equal amounts of the two enantiomers.

A preferred embodiment of the invention is a steroid compound as definedpreviously and further characterised in that R₆ is H, R₇ is a branchedor unbranched C₁₋₃-alkyl, the E-ring is a five or six-membered ringwithout double bonds having a hydroxy at position 22 in Sstereoconfiguration, which configuration in other words is β withrespect to the steroid skeleton according to the meaning common insteroid stereochemistry notification.

Most preferred is the compound according to formula 2, which is (7α,16β, 17α,22S)-7-propyl-16,24-cyclo-19,21-dinorchola-1,3,5(10)-triene-3,17,22-triol,having the code name Org 41621:

Compounds according to this invention enable therefore a more directed,in other words selective modification of the activity of estrogen α or βreceptors in an organism.

A prodrug is defined as being a compound which converts in the body of arecipient to a compound according to formula 1 with R_(E) being aβ-hydroxyl group. Notably, the hydroxy groups at position 3, 17 and onthe E-ring can for example be converted into ethers (alkyl*oxy) oresters such as acyl*oxy, phosphate, sulfate, sulfonate or aromaticcarboxylate, whereby the carbon chain length of the groups denoted withan asterisk (*) is not considered to be sharply delimited. An acyl groupis derived from a linear or branched alkane* and an aromatic carboxylategenerally will comprise a phenyl, pyridinyl or pyrimidyl. The length ofthe alkyl and acyl groups is selected depending on the desiredproperties of the prodrugs, whereby the longer chained prodrugs with forexample lauryl or caproyl chains are more suitable for sustained releaseand depot preparations. It is known that such substituents spontaneouslyhydrolyse or are enzymatically hydrolysed to the free hydroxylsubstituents on the skeleton of the compound. Such prodrugs will havebiological activity comparable to the compounds to which they areconverted in the body of the recipients. The active compound to which aprodrug is converted is called the parent compound. The onset of actionand duration of action as well as the distribution in the body of aprodrug may differ from such properties of the parent compound.

Both for medical therapies as well as for physiological, medical andpharmacological experiments such selective compounds as found now aredesperately needed. It is an aspect of the invention that a compound ofthe invention can be used for therapy by administering the compound to arecipient, being human or an animal, preferably a mammal. The differentdistribution of α and β receptors over different tissues of an organismprovides targets for more selective interference with the functioning ofdifferent tissues. It is known that species dependent, estrogenreceptors α are expressed predominantly in vaginal tissue and livertissue, whereas β receptors are expressed predominantly in prostatetissue, the epithelial cell layer of rat bladder, vascular endothelialsmooth muscle cells and certain brain regions, such as the basalforebrain, neocortex and hippocampus. Tissues wherein both receptors arepresent are for example the pituitary, hypothalamus, thymus, uterus,ovary and bone.

The estrogen-receptor affinity profile of the compounds of the presentinvention, makes them suitable as improved estrogens or anti-estrogensunder diminished estrogen-related side-effects. Thus, the inventiondiscloses a method for selective modification of the activity ofestrogen receptors by bringing a compound of the invention into contactwith estrogen receptors. Such a method can be a treatment of the humanor animal body, but it can also be a non-medical method. The lattermethod can be an experimental method, such as an assay for selectivecompounds or an in vitro laboratory method to obtain information onestrogen receptors or compounds interacting therewith. Preferably, thesecompounds can be used for selective estrogen-receptor α or β relatedcontraceptive, experimental, or medical treatments, such as those fortreatment or prevention of estrogen receptor related disorders,menopausal complaints, osteoporosis, cardiovascular disorders,modulation of pituitary hormone regulation, benign prostate hypertrophy,estrogen dependent tumour control, colon cancer, endometriosis orcentral nervous system disorders. An important common characteristic ofthese selective methods and treatments is that these comprise thebringing of a compound of the invention into contact with estrogenreceptors.

The invention also relates to the use of a compound according to theinvention for the manufacture of a medicament for selective estrogenreceptor related treatment and of a medicament for treatment of estrogenreceptor α related disorders, comprising the administration to a patientof a compound according to the invention (in a suitable pharmaceuticaldosage form). In view of the antagonistic effect of a compound accordingto the invention on the estrogen receptor β the invention also providesfor the manufacture of a medicament for treatment of estrogen receptor βrelated disorders, comprising the administration to a patient of acompound according to the invention (in a suitable pharmaceutical dosageform).

Further, the invention relates to the use of a compound according to theinvention in the manufacture of a medicament having contraceptiveactivity. Thus the invention also pertains to the medical indication ofcontraception, i.e. a method of contraception comprising theadministration to a subject, being a woman or a female animal, of aprogestogen and an estrogen as is customary in the field, wherein theestrogen is a compound according to this invention (in a suitablepharmaceutical dosage form).

Finally the invention relates to the use of a compound according to theinvention for the manufacture of a medicament having selectiveestrogenic activity, such a medicament being generally suitable in thearea of HRT (hormone replacement therapy) having a menopausal complaintsrelieving, in particular, an anti-osteoporose activity.

The dosage amounts of the present compounds will be of the normal orderfor estrogen-related compounds, e.g. of the order of 0.01 to 100 mg peradministration.

To this end dosage units can be prepared containing amounts of acompound of the invention in the same order of magnitude as the aboveindicated treatment doses. Therefore, the present invention also relatesto a pharmaceutical composition comprising a compound of the inventionmixed with one or more pharmaceutically acceptable auxiliaries.

The compounds of the invention may be produced by various methods knownin the art of organic chemistry in general, and especially in the art ofthe chemistry of steroids. See for example: Fried, J. and Edwards, J.A., “Organic Reactions in Steroid Chemistry”, Volumes I and II, VanNostrand Reinhold Company, New York, 1972; and C. Djerassi, “SteroidReactions”, Holden-Day, Inc., San Francisco, 1963.

For the synthesis of a compound according to the invention steroids withan additional 16,17 anellated ring have to be synthesised. Methods to doso have been described in EP 0 869 132. For the compounds of thisinvention an additional hydroxy function must be introduced on theanellated ring. For this purpose it is convenient to carry out theanellation reaction in such a way that suitably located double bondsresult from the synthetic procedure, for example by using a well knownolefin metathesis procedure, whereby transition metal catalysts are usedwhich are derived from e.g. ruthenium, molybdenum, or tungsten, to closea 16α, 17α-bis unsaturated fragment into an unsaturated anellated 5- or6 membered ring. The olefin ring thus synthesised is firststereoselectively epoxidized. This can be done generally with agentslike peracids (preferably in a buffered medium) or catalytic systemsusing metal complexes in the presence of oxidizing agents (like hydrogenperoxide or t-butylhydroperoxide). In the present cases perbenzoic acidwith sodiumbicarbonate buffer generally gives good results. The epoxidescan be almost regioselectively reductively opened with hydride reagentsto the required beta-alcohols. Hydroxy groups may be easily synthesisedas well by application of a hydroboration/oxidation procedure.

In cases where the α-hydroxy compounds are obtained, a Mitsunobuinversion easily leads to the β-alcohols, which are the intended ones.

The hydroxy compounds can be converted into prodrugs, such as alkylethers, acyl esters, carbonates, sulphonates or phosphates, by reactionwith the appropriate alkyl halide or acid chloride as desired.

A pharmaceutical composition comprising one or more compounds accordingto the invention can be prepared with or without combination withpharmaceutically acceptable auxiliaries, such as described in thestandard reference Gennaro et al., Remmington's Pharmaceutical Sciences,(18th ed., Mack publishing Company, 1990, see especially Part 8:Pharmaceutical Preparations and Their Manufacture.). A mixture of one ormore compounds according to the invention and one or morepharmaceutically acceptable auxiliaries may be compressed into soliddosage units, such as pills or tablets, or be processed into capsules orsuppositories. By means of pharmaceutically suitable liquids thecompounds can also be applied as an injection preparation in the form ofa solution, suspension, emulsion, or as a spray, e.g. nasal spray. Thecompounds of the invention may also be included in an implant, a vaginalring, a patch, a gel, and any other preparation for sustained release.For making dosage units, e.g. tablets, the use of conventional additivessuch as fillers or carriers, colorants, polymeric binders and the likeis contemplated. In general any pharmaceutically acceptable additivewhich does not interfere with the function of the active compounds canbe used. Suitable carriers with which the compositions can beadministered include lactose, starch, cellulose derivatives and thelike, or mixtures thereof used in suitable amounts.

EXAMPLES

The routes of synthesis used in the examples are illustrated in theschemes I and II. The numbers used to identify the compounds are definedby the structural formulas in these schemes.

Compound 2

To a solution of 5.65 ml of vinyltributyltin in 50 ml of dry THF wasadded at −50° C. dropwise 12 ml of 1.6 M BuLi in hexane. After stirringfor an additional 0.5 hr a solution of 6.2 gr of16α-allyl,7α-ethylestrone-3-O-methylether (1) in 20 ml of dry THF wasadded at −50° C. Upon stirring for an additional ½ h the mixture waspoured into sat.NH₄Cl and extracted with ethylacetate. Concentration ofthe organic phase followed by silicagel chromatography gave 4.2 g of 2as an oil,

R_(f) 0.47 (toluene/ethyl acetate 95/5), for 1 R_(f) 0.65. NMR (CDCl₃) δ5.80 (m, 1, CH allyl), 6.07 (m, 1, CH vinyl) 0.98 (s, CH₃), 0.93 (t, 3,ethyl), 3.78 (s, 3, CH₃).

Compound 3

To a solution of 4.2 g of 2 in 80 ml of methylenechloride was added 0.32g of benzylidenetriscyclohexylphosphinoruthenium dichloride (Grubbsmetathesis catalyst). After stirring for 1 hr an additional portion of0.3 g of catalyst was added. After completion of the reaction (2 h) themixture was concentrated and the residue purified by columnchromatography, to provide 3.5 g of 3, R_(f) 0.29 (heptane/ethyl acetate8/2, for 2 R_(f) 0.55). NMR (CDCl₃) δ5.72 (m, CH═) 6.02 (m, 1, C═) 0.99(s, 3, CH₃), 0.89 (t, 3, CH3) 3.77 (OCH₃) 0.92 (m, 3, CH₃).

Compound 4

A mixture of 0.4 g of steroid 3 and 0.5 g of NaHCO₃ in 12 ml ofmethylenechloride was treated with 0.34 g of m-Cl-perbenzoic acid. Afterstirring for several hours at ambient temperature the reaction wasdiluted with water, and treated with sodiumthiosulphate solution todestroy residual peroxide. The organic material was extracted into ethylacetate and finally purified by chromatography, to provide 110 mg of thedesired β-epoxide 4; R_(f) 0.50 (toluene-acetone 9/1); NMR(CDCl₃) δ 0.98(t, 3, CH₃), 0.92 (t, 3, CH₃), 3.65+3.70 (2×m, epoxide CH's).

Compound 5

A solution of 80 mg of steroid 4 in 3 ml of THF was refluxed with 10 mgof LiAlH₄. After 2 h starting material had disappeared. The mixture wasquenched by addition of 30 μl of sat. Na₂SO₄ solution and 0.20 g ofNa₂SO₄, stirred for 15 min and filtered over Celite. The filtrate wasconcentrated and the residue passed over a short silica column, to give55 mg of 5; R_(f) 0.24 (toluene-acetone 9/1); NMR(CDCl₃) δ 4.04 (m, 1,CHOH), 3.78 (s, 3, OCH₃) 2.85 (m, 2, CH₂ at C6), 0.92 (s, 3, CH₃).

Compound 6

To a solution of sodiumethanethiolate (prepared from 0.7 ml ofethanethiol and 0.27 g of a 60% NaH dispersion) in 9 ml of DMF was added120 mg of steroid 5. The mixture was refluxed for 3 h. Then the reactionwas poured into water and extracted with ethyl acetate. Chromatographyof the organic material provided 80 mg of 6, Mp 224-226; R_(f) 0.30(toluene-acetone 8/2); NMR(CDCl₃) δ 4.00 (m, 1, CHOH), 7.12 (ar H1),6.20 (ar H2), 6.54 (ar H4).

Compound 7

To a solution of 2.7 g of steroid 3 in 20 ml of methylenechloride and 3ml of pyridine, was added at 0° C. 1.9 ml of trimethylsilylchloride.After stirring for ½ h the reaction was poured into water and extractedwith ethyl acetate, to provide 3.3 g of essentially pure silyloxyderivative 7, R_(f) 0.8 (heptane-acetone 8/2); NMR(CDCl₃) δ 0.03 (s, 9,TMS), 5.68, 5.91 (2×s, CH olefin).

Compound 8

A solution of 3.2 g of 7 in 25 ml of dry THF was treated at 0° C. with asolution of 2.2 ml borane-dimethylsulfide complex in 20 ml of THF. Thereaction was subsequently stirred for 2 hr at 45° C. Excess reagent wasdestroyed by careful addition of 4.5 ml of abs.ethanol, followed by 11ml of 2N NaOH and 7.7 ml of 30% hydrogenperoxide. The reaction wasstirred overnight, diluted with water and extracted with ethyl acetate.The crude product thus obtained was purified by column chromatographyand yielded 1.7 g of the desired α-alcohol 8. R_(f) 0.36(heptane-acetone 8/2); NMR(CDCl₃) δ 4.42 (m, 1, CHOH), 0.12 (s, 9, TMS),0.79 (s, 3, CH3).

Compound 9

A solution of 1.6 g of 8 and 1.3 g of triphenylphosphine in 60 ml oftoluene was treated with 0.84 g of p-nitrobenzoic acid and 0.8 ml ofdiethylazodicarboxylate at 0° C. After stirring for 1 h the reaction wascomplete. The mixture was poured onto sat.aq. NaHCO₃ solution andextracted with ethyl acetate. Chromatografic purification gave 2.9 g ofβ-nitrobenzoate, R_(f) 0.64 (heptane-acetone 8/2); NMR(CDCl₃) δ 5.34 (m,1, CHOC(O)Ar), 0.91 (s, 3, CH₃), 0.95 (t, 3, CH₃), 3.80 (s, 3, OCH₃).This material was dissolved in a mixture of 40 ml of THF-methanol (1/1v/v) and treated with 4 ml of 2N NaOH solution. After stirring for 15min the reaction was poured into water and the product extracted intoethyl acetate. After passing through a short silica column 1.3 g ofβ-alcohol 9 was obtained. R_(f) 0.64 (heptane-acetone 8/2); NMR(CDCl₃) δ4.31 (m, 1, CHOH).

Compound 10

A solution of 1.3 g of 9 in 30 ml of acetone was treated with 2 ml of 2NHCl. After 1 h the mixture was neutralized by addition of saturatedNaHCO₃ and concentrated. The residue was diluted with water andextracted with ethyl acetate, to provide 1.0 g of 10, R_(f) 0.10(heptane-acetone 8/2); NMR(CDCl₃) δ 0.90 (t, 3, CH₃) 0.95 (s, 3, CH₃),4.48 (m, 1, CHOH).

Compound 11

To a solution of sodiumethanethiolate (prepared from 0.7 ml ofethanethiol and 0.3 g of a 60% NaH dispersion) in 9 ml of DMF was added120 mg of steroid 10. The mixture was refluxed for 3 h. Then thereaction was poured into water and extracted with ethyl acetate.Chromatography of the organic material provided 80 mg of 11, Mp 143-145°C. (ethanol-water); R_(f) 0.41 (toluene-acetone 7/3); NMR(CDCl₃) δ 4.45(m, 1, CHOH), 0.93 (s, 3, CH₃) 0.90 (t, 3, CH₃), 6.62+7.10 (AB, 2, H1,2), 6.53 (d, 1, H4).

Compound 13

To a solution of 29 ml of 1M allylmagnesium bromide in ether was added80 ml of dry THF. At −50° C. was added 10 gr of 7α-propyl,16α-allylestrone-3-O-benzylether 12 in 40 ml of THF. After stirring for½ h the mixture was allowed to come to room temperature and poured into300 ml of sat aq NH₄Cl. The product was extracted with ethyl acetate andpurified, by chromatography over silica gel to remove stereoisomersproviding 7.2 g of the desired 16α,17α-diallyl derivative 13. R_(f) 0.26(heptane-ethylacetate 9/1); 17β-allyl isomer R_(f) 0.45. NMR(CDCl₃) δ0.88 (t, 3, CH₃), 0.96 (s, 3, CH₃), 6.08 (m, 1, CH allyl), 5.80 (m, 1,CH allyl), 4.95-5.20 (m, 4, 2× CH₂ allyl) 5.02 (CH₂OBz).

Compound 14

A portion of 40 mg of benzylidenetriscyclohexylphosphinorutheniumdichloride (Grubbs metathesis catalyst) was added to a solution of 450mg of 13 in 10 ml of methylenechloride. After stirring for 1 h anadditional 400 mg of catalyst were added. After 2 h, the solvent wasremoved and replaced by 20 ml of toluene and the mixture was stirredwith 5 g of basic alumina at 60° C. to absorb the catalyst. Afterfiltration over Celite and washing with toluene and ethyl acetate, 400mg of almost pure 14 was obtained; R_(f) 0.34 (heptane-ethylacetate8/2); R_(f) 13 0.45. NMR(CDCl₃) δ 0.02 (s, 2, CH₂O), 5.97 (s, 2, CH═CH),0.97 (s, 3, CH₃)

Compound 15

To a solution of 340 mg of 14 in 0.5 ml of methylenechloride was added 8μl of pyridine, 0.14 ml of 30% aq H₂O₂, followed by 2 mg ofmethyltrioxorhenium. After stirring for 1 h the epoxidation wascomplete, leading predominently to the desired β-epoxide as well as someα-isomer. The mixture was poured onto water and sat. Na₂S₂O₃ andextracted with ethylacetate. Chromatography provided 230 mg of 15 and 80mg of the undesired α-epoxide. R_(f) 0.36 (toluene-ethylacetate 95/5;for reference: R_(f) compound 14: 0.39). NMR(CDCl₃) δ 3.31, 3.38 (m, 2,CH(O)CH).

Compound 16

A mixture of 4.2 g of 15 and 350 mg of LiAlH₄ in 20 ml of dry THF wasrefluxed for 2 h. Then the mixture was cooled and subsequently treatedwith 1 ml of sat aq. Na₂SO₄, 28 ml of ethyl acetate and 8 g of Na₂SO₄.After stirring for ½ h at ambient temperature the reaction was filteredover Celite and the filtrate concentrated and chromatographed, toprovide 3.4 g of 16, Mp 147-148° C., R_(f) 0.20 (toluene-ethylacetate8/2; R_(f) 23 0.55). NMR(CDCl₃) δ 4.25 (broad s, 1, CHOH), 0.90 (s, 3,CH₃), 0.86 (t, 3, CH₃), 5.02 (s, 2, OCH₂Ar).

Compound 17; (=7α, 16β, 17α,22S)-7-propyl-16,24-cyclo-19,21-dinorchola-1,3,5(10)-triene-3,17,22-triol

A solution of 3.3 g of 16 in 200 ml of ethanol was hydrogenated in thepresence of 300 mg of 5% Pd/C. After completion of the reaction thecatalyst was filtered over Celite, and the solvent concentrated. Theresidue was triturated with ether, and then with water, to provide 1.7 gof 17; Mp 146-147° C., R_(f) 0.53 (toluene-ethylacetate 1/1; R_(f) 160.60). NMR(CDCl₃) δ 4.26 (broad s, 1, CHOH) 7.14, 6.62 AB, 2, H1, H2),6.54 (d, H4).

Tests for Estrogenic Activity In Vitro

Compounds are tested for their estrogen receptor activity in a bindingassay and in a transactivation assay using the human estrogen receptor αor β.

Competitive binding to cytoplasmic human estrogen receptor α or β fromrecombinant Chinese hamster ovary (CHO) cells is used to estimate therelative binding affinity (=RBA)(potency ratio) of a test compound ascompared with (17β)-estradiol (E₂) for estrogen receptors α or β presentin the cytosol of recombinant CHO cells, stably transfected with thehuman estrogen receptor α (hERα) or β receptor (hERβ).

The estrogenic and antiestrogenic activity of compounds is determined inan in vitro bioassay with recombinant Chinese hamster ovary (CHO) cellsstably co-transfected with the human estrogen receptor α (hERα) or β(hERβ), the rat oxytocin promoter (RO) and the luciferase reporter gene(LUC). The estrogenic agonistic transactivation (potency ratio) of atest compound to stimulate the transactivation of the enzyme luciferasemediated via the estrogen receptors hERα or hERβ is compared with thestandard estrogen estradiol. The antiestrogenic activity (potency ratio)of a test compound to inhibit the transactivation of the enzymeluciferase mediated via the estrogen receptors hERα or hERβ by the(17β)-estradiol is compared with the standard ICI 164.384(=(7α,17β)-N-butyl-3,17-dihydroxy-N-methylestra-1,3,5(10)-triene-7-undecanamide).

Results:

Formula 3

6-ring Agonist Com- (n = 1) or Binding- Trans- selec- pound as 5-ringassay activation tivity Form. 3 R₇ R₁₁ (n = 0) Erα/Erβ Erα/Erβ Erα/Erβ AH H 6-ring 8.6/0.3 5.5/0.2 27 B CH₃ H 6-ring 20/5.2 16/<0.1 >160 C(=11)C₂H₅ H 5-ring 18/14 12/<0.1 >120 D C₂H₅ H 6-ring 17/8.7 11.5/<0.1 >115E(=17¹⁾) C₃H₇ H 6-ring 42/13.9 26/<0.1 >260 F H C₃H₇ 5-ring 16.3/0.411/0.2 55 ¹⁾17 is Org 41621

Compound 17 has in the antagonist assays a selectivity ratio Erα/Erβ of<0.1/67.

A compound of the prior art according to formula 3, but without22-hydroxy and having R₇ α-propyl, R₁₁ hydrogen and 6-membered E-ring,which is named (7α, 16β,17α)-7-propyl-16,24-cyclo-19,21-dinorchola-1,3,5(10)-triene-3,17-diol,has a ERα/ERβ ratio in the binding assay of 15/7 and in the agonisttransactivation assay 0.3/<0.1.

Test for Prevention of Ovariectomy-Induced Bone Loss in Rats(Anti-Osteoporosis Test).

Introduction

Ovariectomy of rats induces bone loss, which is due to estrogendeficiency. Administration of estrogenic compounds prevents this effect.The test is used to evaluate a compound for anti-osteoporotic activityin ovariectomised (OVX) rats. The effect on bone mass can be evaluatedby peripheral Quantitative Computed Tomography (pQCT), or byquantitative analysis of X-ray pictures. Plasma osteocalcin and urinarydeoxypyridinoline, calcium and phosphate gives information on bonemetabolism. Increase of uterine weight and decrease of body and thymusweight reflect estrogenic effect.

Test Animal

Mature virgin 225-250 g female Wistar rats. Strain: Hsd/Cpd:Wu, SPF-bredby Harlan, CPB, Zeist, The Netherlands.

Experiment

On day 1 of the experiment the rats are weighed and distributed over thecages in order of bodyweight, whereby the rat with the smallestbodyweight is placed in the first cage and the heaviest rat in the finalcage. Treatments are randomised over the rats.

Sham-operation and ovariectomy are performed under ether anaesthesia.After recovery from the anaesthesia, within 24 h, vehicle, referencecompound or test compound is administered once or twice daily for 4weeks. During this period the rats are weighed weekly. After 4 weeksautopsy was performed. At autopsy the rats are anaesthetised with etherand blood is collected from the abdominal aorta. Both femora, thevertebrae L1L2L3L4 (optionally), uterus, thymus, liver, kidneys,adrenals, thyroid, and pituitary gland are dissected out. Measurement ofbone mineral density and geometry of the right femur is performed bypQCT on the day of autopsy on fresh tissue. Trabecular bone mineraldensity of the metaphyseal part of the femur (FBMDDIS mg/cm³) ismeasured with a pQCT (peripheral Quantitative Computed Tomographymachine; XCT 960A, Stratec, Birkenfeld, Germany).

Interpretation of Results

Ovariectomy causes a statistically significant decrease in distal bonedensity and trabecular bone volume of the femur and a statisticallysignificant increase in plasma osteocalcin and urinary deoxypyridinolinelevels (P≦0.05, 2 way ANOVA).

Test compounds are considered to be active when mean bone density valuesof the distal femur are significantly increased as compared to theovariectomised control group. Effects of compounds on urinarydeoxypyridinoline levels reflects an effect on bone resorption, effectson plasma osteocalcin levels reflects an effect on bone turnover and mayhelp to understand the mechanism of action.

The minimal active dose (MAD) is the dose where a mean proportionaldifference in trabecular bone mineral density between 40 and 60% isreached.

REFERENCES

-   Wronski T. J. and Yen C. F.: The ovariectomised rat as an animal    model for postmenopausal bone loss. Cells and Materials, Supp. 1    (1991): 69-76.-   Yamazald I. and Yamaguchi H.: Characteristics of an ovariectomised    osteopenic rat model. J. Bone Min. Res. 4 (1989): 12-22.-   Ederveen A. G. H. and Kloosterboer H. J.: Tibolone, a steroid with a    tissue-specific hormonal profile, completely prevents    ovariectomy-induced bone loss in sexually mature rats. J. Bone &    Mineral Research Vol 14, pp 1963-1970, 1999.    Result:

Compound 17 (Org 41621): Osteoporosis test oral 30 μg/kg. Prior artcompound (7α, 16β,17α)-7-propyl-16,24-cyclo-19,21-dinorchola-1,3,5(10)-triene-3,17-diol:190 μg/kg per oral.

Test for Strogenic Activity In Vivo

In vivo estrogenic activity was determined by means of the Allen Doisytest, described in F. Allen, L. A. Doisy, J. Amer. Med. Assoc., 81,819-821 (1923)

Result:

Compound 17 (Org 41621): Allen Doisy sc 5 μg/kg, oral 30 μg/kg. Priorart compound (7α, 16β,17α)-7-propyl-16,24-cyclo-19,21-dinorchola-1,3,5(10)-triene-3,17-diol:Allen Doisy sc 24 μg/kg, oral 125 μg/kg.

1. A compound according to formula 2:


2. A steroid compound having formula 1:

wherein R₇ is H, R₁₁ is C₃H₇ and n is
 0. 3. A pharmaceuticalcomposition, comprising: the steroid compound according to claim 1 and apharmaceutically acceptable auxiliary.
 4. A pharmaceutical composition,comprising: the steroid compound according to claim 2 and apharmaceutically acceptable auxiliary.